Detection and amplification of ligands

ABSTRACT

Devices and systems for the detection of ligands comprising at least one receptor and an amplification mechanism comprising a liquid crystalline, where an amplified signal is produced as a result of receptor binding to a ligand are provided. Also provided are methods for the automatic detection of ligands.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S. Ser.No. 09/633,327, filed Aug. 7, 2000, which is a continuation of U.S. Ser.No. 09/095,196, filed Jun. 10, 1998, now U.S. Pat. No. 6,171,802.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention generally relates to the detection of aligand by a receptor. More specifically, the present invention relatesto highly specific receptors and the incorporation of these receptorsinto an amplification mechanism comprising a liquid crystalline materialfor the rapid and automatic detection of the ligand, such asmicroorganisms and products of microorganisms, such as pathogens and/ortheir toxins.

BACKGROUND OF THE INVENTION

[0003] The detection of a ligand by a receptor (for example, detectionof a pathogenic agent such as a microbe or toxin by an antibody; ordetection of an antibody in blood by another antibody; or binding of achemical toxin, such as nerve gas, to its receptor) is important in thediagnosis and treatment of individuals exposed to disease-causingagents. Early detection of pathogenic agents can be a great benefit ineither disease prophylaxis or therapy before symptoms appear or worsen.

[0004] Every species, strain, or toxin of a microbe contains uniqueinternal and external ligands. Using molecular engineering and/orimmunological techniques, receptor molecules, such as antibodies, can beisolated that will bind to these ligands with high specificity. Methodshave also been developed where receptors, such as antibodies, are linkedto a signaling mechanism that is activated upon binding. Heretofore,however, no system has been developed that can automatically detect andamplify a receptor signal coming from the binding of a single or a lownumber of ligands in near real time conditions. Such a system isimperative for rapid and accurate early detection of ligands.

[0005] Many available diagnostic tests are antibody based, and can beused to detect either a disease-causing agent or a biologic productproduced by the patient in response to the agent. There are currentlythree prevailing methods of antibody production for recognition ofligands (antigens): polyclonal antibody production in whole animals withrecognition for multiple epitopes, monoclonal antibody production intransformed cell lines with recognition for a single epitope (afterscreening), and molecularly engineered phage displayed antibodyproduction in bacteria with recognition of a single epitope (afterscreening). Each of these receptor systems is capable of binding andidentifying a ligand, but the sensitivity of each is limited by theparticular immunoassay detection system to which it is interfaced.

[0006] Immunoassays, such as enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), and radioimmunoassay (RIA), are well known forthe detection of antigens. The basic principle in many of these assaysis that an enzyme-, chromogen-, fluorogen-, orradionucleotide-conjugated antibody permits antigen detection uponantibody binding. In order for this interaction to be detected as acolor, fluorescence, or radioactivity change, significant numbers ofantibodies must be bound to a correspondingly large number of antigenepitopes.

[0007] Thus, there is a need for a system that rapidly, reliably, andautomatically detects ligands, especially when present in very smallquantities and consequently provides a measurable signal in near realtime conditions.

SUMMARY OF THE INVENTION

[0008] It is, therefore, an object of the present invention to provide adevice, system, and method that will detect a ligand with highsensitivity and high specificity in near real time.

[0009] It is another object of the present invention to provide adevice, system, and method that will amplify a signal produced by thebinding of a ligand to a receptor.

[0010] It is a further object of the present invention to provide adevice and system that will distort a surrounding liquid crystallinematerial upon the binding of a ligand to a receptor.

[0011] In general, the present invention provides a system for thedetection and amplification of ligands, such as pathogenic agents,comprising at least one receptor and an amplification mechanismcomprising a liquid crystalline material coupled to that receptor,wherein an amplified signal is produced as a result of the receptorbinding the ligand.

[0012] In one embodiment, the present invention provides a device forthe detection of ligands comprising at least one substantially sphericalsubstrate; at least one receptor attached to said spherical substrate,wherein said at least one receptor is capable of binding to a ligand toform a receptor-ligand complex and wherein the formation of saidreceptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation.

[0013] In another embodiment, the present invention also provides amethod for detecting ligands comprising providing a device capable ofdetecting ligands, said device comprising at least one substantiallyspherical substrate, at least one receptor attached to said sphericalsubstrate, wherein said at least one receptor is capable of binding to aligand to form a receptor-ligand complex and wherein the formation ofsaid receptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism comprises a liquid crystalline material andamplifies said signal upon receptor-ligand complex formation; exposing asample containing at least one ligand to at least one substrate;allowing said receptor to interact with said at least one ligand to format least one receptor-ligand complex, and measuring the signal generatedby said receptor-ligand complex formation.

[0014] In another embodiment, the present invention further provides adevice for the detection of ligands comprising: at least onesubstantially spherical substrate coated with a receptor-bindingmaterial; at least one receptor attached to said coated sphericalsubstrate, wherein said at least one receptor is capable of binding to aligand to form a receptor-ligand complex and wherein the formation ofsaid receptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation.

[0015] The present invention also provides a method for detectingligands comprising: providing a device capable of detecting ligands,said device comprising at least one substantially spherical substratecoated with a receptor-binding material; at least one receptor attachedto said coated spherical substrate, wherein said at least one receptoris capable of binding to a ligand to form a receptor-ligand complex andwherein the formation of said receptor-ligand complex produces a signal;and an amplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation; exposing a sample containing at leastone ligand to at least one of said substrate; allowing said receptor tointeract with said at least one ligand to form at least onereceptor-ligand complex; and measuring the signal produced by saidreceptor-ligand complex formation.

[0016] The present invention further provides a device for the detectionof ligands comprising: a substantially planar substrate, wherein saidsubstrate is electrically charged; at least one receptor attached tosaid charged substrate, wherein said at least one receptor is capable ofbinding to a ligand to form a receptor-ligand complex and wherein theformation of said receptor-ligand complex produces a signal; and anamplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation.

[0017] The present invention further includes a method for detectingligands comprising: providing a device capable of detecting ligands,said device comprising at least one electrically charged substantiallyplanar substrate; at least one receptor attached to said substrate,wherein said at least one receptor is capable of binding to a ligand toform a receptor-ligand complex and wherein the formation of saidreceptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation; exposing a sample containing at least one ligand tosaid substrate; allowing said receptor to interact with said at leastone ligand to form at least one receptor-ligand complex; and measuringthe signal produced by said receptor-ligand complex formation.

[0018] The present invention further provides a device for the detectionof ligands comprising: a substantially planar substrate coated with areceptor-binding material; at least one receptor attached to said coatedsubstrate, wherein said at least one receptor is capable of binding to aligand to form a receptor-ligand complex and wherein the formation ofsaid receptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation.

[0019] The present invention also provides a method for detectingligands comprising: providing a device capable of detecting ligands,said device comprising substantially planar substrate coated with areceptor-binding material; at least one receptor attached to said coatedsubstrate, wherein said at least one receptor is capable of binding to aligand to form a receptor-ligand complex and wherein the formation ofsaid receptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation; exposing a sample containing at least one ligand tosaid substrate; allowing said receptor to interact with said at leastone ligand to form at least one receptor-ligand complex; and measuringthe signal produced by said receptor-ligand complex formation.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1A is a schematic representation of the lamellar structure ofa lyotropic liquid crystal formed by alternating layers of water andbiphilic molecules.

[0021]FIG. 1B is a schematic representation of the amplificationmechanism with a receptor inserted into the lyotropic liquid crystal.

[0022]FIG. 1C is a schematic representation of the amplificationmechanism with the specific ligand bound to its receptor causingdeformation of the liquid crystal and alteration of the transmission ofpolarized light.

[0023]FIG. 2A is a representation of a non-porous (solid) sphericalsubstrate having a plurality of receptors attached to the outer surfaceof the sphere.

[0024]FIG. 2B is a representation of a porous spherical substrate havinga plurality of receptors attached to the outer surface of the sphere andwithin the pores of the sphere.

[0025]FIG. 2C is a representation of a non-porous (solid) sphericalsubstrate having a plurality of receptors attached to the outer surfaceof the sphere with ligand bound to a portion of the receptors.

[0026]FIG. 2D is a representation of a porous spherical substrate havinga plurality of receptors attached to the outer surface of the sphere andwithin the pores of the sphere with ligand bound to a portion of thereceptors.

[0027]FIG. 3A is a representation of, a non-porous (solid) sphericalsubstrate having a plurality of receptors attached to the outer surfaceof the sphere showing the liquid crystalline material orientation aboutthe receptor-bound sphere.

[0028]FIG. 3B is a representation of, a porous spherical substratehaving a plurality of receptors attached to the outer surface of thesphere and within the pores of the sphere showing the liquid crystallinematerial orientation about the receptor-bound sphere.

[0029]FIG. 3C is a representation of a non-porous (solid) sphericalsubstrate having a plurality of receptors attached to the outer surfaceof the sphere with ligand bound to a portion of the receptors showingthe change in liquid crystalline material orientation about the spherewhen ligand is bound.

[0030]FIG. 3D is a representation of a porous spherical substrate havinga plurality of receptors attached to the outer surface of the sphere andwithin the pores of the sphere with ligand bound to a portion of thereceptors showing the change in liquid crystalline material orientationabout the sphere when ligand is bound.

[0031]FIG. 4A is a graph showing the number of light transmissivemicrodomains in the neutral grey liquid crystalline material using (a)polycarboxylate microspheres coated with anti-E.coli antibody and (b)polycarboxylate microspheres coated with Bovine Serum Albumin (BSA). Theopen circles (o) represent the number of light transmissive microdomainsin the neutral grey liquid crystalline material using polycarboxylatemicrospheres coated with anti-E.coli antibody, and the filled in circles()represents the number of light transmissive microdomains in theneutral grey liquid crystalline material using polycarboxylatemicrospheres coated with BSA.

[0032]FIG. 4B is a graph showing the number of light transmissivemicrodomains in the neutral grey liquid crystalline material using (a)polystyrene microspheres coated with anti-E.coli antibody and (b)polystyrene microspheres coated with Bovine Serum Albumin. The opencircles (o) represent the number of light transmissive microdomains inthe neutral grey liquid crystalline material using polystyrenemicrospheres coated with anti-E.coli antibody, and the filled in circles()represents the number of light transmissive microdomains in theneutral grey liquid crystalline material using polystyrene microspherescoated with (BSA).

[0033]FIG. 5A is a graph showing the number of light transmissivemicrodomains in the disodium cromoglycate liquid crystalline materialusing (a) polycarboxylate microspheres coated with anti-E.coli antibodyand (b) polycarboxylate microspheres coated with Bovine Serum Albumin.The open circles (o) represent the number of light transmissivemicrodomains in the disodium cromoglycate liquid crystalline materialusing polycarboxylate microspheres coated with anti-E.coli antibody, andthe filled in circles ()represents the number of light transmissivemicrodomains in the disodium cromoglycate liquid crystalline materialusing polycarboxylate microspheres coated with BSA.

[0034]FIG. 5B is a graph showing the number of light transmissivemicrodomains in the disodium cromoglycate liquid crystalline materialusing (a) polystyrene microspheres coated with anti-E.coli antibody and(b) polystyrene microspheres coated with Bovine Serum Albumin. The opencircles (o) represent the number of light transmissive microdomains inthe disodium cromoglycate liquid crystalline material using polystyrenemicrospheres coated with anti-E.coli antibody, and the filled in circles()represents the number of light transmissive microdomains in thedisodium cromoglycate liquid crystalline material using polystyrenemicrospheres coated with BSA.

[0035]FIG. 6A is a representation of a planar substrate having aplurality of receptors attached to one surface of the substrate andwithout ligand bound to the receptors.

[0036]FIG. 6B is a representation of a substantially planar substratehaving a plurality of receptors attached to one surface of the substrateand with some ligands bound to a portion of the receptors.

[0037]FIG. 7A is a representation of a planar substrate having aplurality of receptors attached to one surface of the substrate withoutligand bound to the receptors showing the liquid crystalline materialorientation when ligand is not bound to receptor.

[0038]FIG. 7B is a representation of a planar substrate having aplurality of receptors attached to one surface of the substrate withsome ligands bound to a portion of the receptors showing the change inliquid crystalline material orientation when ligand is bound toreceptor.

DETAILED DESCRIPTION OF THE INVENTION

[0039] In the present invention, ligand-specific receptors areinterfaced with an amplification mechanism such that a receptor-ligandinteraction forms birefringent receptor-ligand aggregates and/or changesthe conformation of the receptor and produce a light transmissivesignal. Amplification preferably occurs through a birefringent shiftthat can be photometrically detected. The detected signal may then beelectronically amplified to automate the system.

Ligand Detection Component

[0040] Any receptor, such as antibodies or biologic/biologicallyengineered receptors for ligands, can be incorporated into the device aslong as binding of the ligand to the receptor causes a detectable ligandaggregation and/or distortion (change in conformation) of the receptor.For example, any type of monospecific antibody (polyclonal, monoclonal,or phage displayed) can effectively function as a receptor and, thus,each of those antibody types will be described in the followingparagraphs. Although phage-displayed antibodies can be expeditiouslymodified for identification of new ligands and are used as receptorexamples in this patent application, any physically-distortablereceptor-ligand interaction is appropriate for the detection component.

[0041] Polyclonal antibodies: Antibody-based antigen detection has beenexploited for several decades. Injection of a purified ligand (antigen)into a host animal stimulates the immune system to produce an array ofantibodies against various reactive sites on the antigen. Since severallymphocytes are responding to different antigenic epitopes, amulti-specific antibody cocktail (polyclonal) is created and can bepurified for antigen detection.

[0042] Monoclonal antibodies: Antibody-producing spleen cells (Blymphocytes) are fused with immortalized myeloma cells to createhybridomas which provide nearly infinite quantities of antibody with asingle, defined specificity. Interstrain and even interspecies hybridsof these ‘monoclonal’ antibodies can be generated through geneticengineering techniques. These highly specific antibodies havesignificant therapeutic potential, as evidenced by the U.S. Food andDrug Administration's approval of the use of mouse-human chimericantibodies for treatment of selected diseases.

[0043] Phage-displayed mono-specific antibodies: Phage-displayedtechniques will be used to isolate single chain chimeric antibodies tovarious pathogenic agents. The genomic DNA of the B lymphocyte containsthe code to produce an antibody to virtually all possible ligands(antigens). In a phage displayed antibody system (PDA), DNA encoding asingle chain chimnera of the native antibody's hypervariableligand-binding region is synthesized by joining DNA encoding an antibodyheavy chain and DNA encoding an antibody light chain and insertingtherebetween DNA encoding a linker region. The desired amino acidsequence of the linker region depends on the characteristics requiredfor any given amplification mechanism. The linker region may have to beable to interact and/or bond to a protein or other substance. Therefore,the polypeptide sequence may have to have, for example, a particularconformation, specifically placed functional groups to induce ionic orhydrogen bonds, or a hydrophobicity that is compatible with theamplification mechanism. Regardless of the type of amplificationmechanism, however, the linker region plays a critical role ininterfacing the amplification mechanism to the receptor.

[0044] The DNA, preferably human or mouse, encoding the single chainchimeric antibody is cloned into a bacteriophage (phage) vector usingwell-known techniques (Marks et al., J. Mol. Bio. Vol. 222:581 (1991);Griffiths et al., EMBO J. 12:725 (1993); and Winters et al., Ann. Rev.Immunol. 12:433 (1994)), incorporated herein by reference. The singlechain chimeric antibodies then become displayed on the surface of afilamentous phage with the hypervariable antigen-binding site extendedoutward.

[0045] After the addition of ligands, phage that are reactive againstnon-targeted ligands are subtracted from the phage library using knowntechniques (Marks et al., J. Mol. Bio. Vol. 222:581 (1991); Griffiths etal., EMBO J. 12:725 (1993); and Winters et al., Ann. Rev. Immunol.12:433 (1994)), incorporated herein by reference. The remaining phageare reacted with their specific ligand and phage reactive with thatspecific ligand eluted. Each of these phage are then isolated andexpressed in a bacterial host, such as Escherichia coli (E. coli) toproduce a large quantity of phage containing the desiredsurface-displayed antibody. Each of the aforementioned methods relatingto synthesizing and cloning DNA, subtracting phages, isolating andexpressing phages and recovering viral DNA are well known and fullydescribed by Marks et al., J. Mol. Biol. (1991); Griffiths et al., EMBOJ. 12:725 (1993); and Winters et al., Ann. Rev. Immunol. 12:433 (1994),all of which are incorporated herein by reference.

Amplification Component

[0046] An amplification mechanism including liquid crystalline materialis utilized to amplify a ligand-receptor complex, thereby detecting thepresence of ligands in a sample.

[0047] A liquid crystal is a state of matter in which molecules exhibitsome orientational order but little positional order. This intermediateordering places liquid crystals between solids (which possess bothpositional and orientational order) and isotropic fluids (which exhibitno long-range order). Solid crystal or isotropic fluid can be caused totransition into a liquid crystal by changing temperature (creating athermotropic liquid crystal) or by using an appropriate diluting solventto change the concentration of solid crystal (creating a lyotropicliquid crystal). Both thermotropic and lyotropic liquid crystals can beused as the amplification mechanism of the device of the presentinvention. In one embodiment, chromonic lyotropic liquid crystallinematerial are used as the amplification component of the device of thepresent invention.

[0048] Among these non-surfactant lyotropic liquid crystals areso-called lyotropic chromonic liquid crystals (LCLCs). The LCLC familyembraces a range of dyes, drugs, nucleic acids, antibiotics,carcinogens, and anti-cancer agents. For a review of lyotropic chromonicliquid crystals see J. Lydon, Chromonics, in: Handbook of LiquidCrystals, Wiley-VCH, Weinheim, vol. 2B, p. 981 (1998). The LCLCs arefundamentally different from the better known surfactant-based lyotropicsystems. Without limitation, one difference is that LCLC molecules aredisc-like or plank-like rather than rod-like. The polar hydrophilicparts form the periphery, while the central core is relativelyhydrophobic. This distinction creates a range of different orderedstructures. Individual disc-like molecules may form cylindricalaggregates in water. The LCLCs are assumed to be formed by elongatedaggregates, lamellar structures, and possibly by aggregates of othershapes.

[0049] As seen in FIG. 1A, most lyotropic liquid crystals, designatedgenerally by the numeral 1, are formed using water 2 as a solvent forbiphilic molecules 3, for example, molecules which possess polar(hydrophilic) parts 4 and a polar (hydrophobic) parts 5. When water 2 isadded to biphilic molecules 3, a bilayer 6 forms as the hydrophobicregions coalesce to minimize interaction with water 2 while enhancingthe polar component's interaction with water. The concentration andgeometry of the specific molecules define the supramolecular order ofthe liquid crystal. The molecules can aggregate into lamellae as well asdisk-like or rod-like micelles, or, generally, aggregates of anisometricshape. These anisometric aggregates form a nematic, smectic, columnarphase, of either non-chiral or chiral (cholesteric phase) nature. Forexample, the molecules form a stack of lamellae of alternating layers ofwater and biphilic molecules, thus giving rise to a lamellar smecticphase.

[0050] Lyotropic liquid crystals are usually visualized as orderedphases formed by rod-like molecules in water. A fundamental feature ofthe surfactant molecules is that the polar hydrophilic head group has anattached flexible hydrophobic tail. There is, however, a variety ofother lyotropic systems that are not of the surfactant type, but whichcan also be successfully used in the present invention.

[0051] Liquid crystalline phases are characterized by orientationalorder of molecules or their aggregates. In the uniaxial liquid crystalphases such as nematic and smectic A, the average direction oforientation of the molecules or aggregates is described by a unitvector, called the director and denoted n. Generally, the two oppositedirections of the director are equivalent, n=−n. In the uniaxial phases,the director is simultaneously the optical axis of the medium. Anoptically uniaxial liquid crystalline medium is birefringent. A uniaxialbirefringent medium is characterized by two optical refractive indices:an ordinary refractive index “n_(o)” for an ordinary wave and anextraordinary refractive index “n_(e)” for an extraordinary wave.

[0052] When the liquid crystal is viewed between two crossed polarizers,the appearing texture and the intensity of transmitted light aredetermined by orientation of the optical axis (director) with respect tothe polarizers and other factors, as clarified below.

[0053] Consider, as an example, a nematic slab sandwiched between twoglass plates and placed between two crossed polarizers. We follow thedescription given by M. Kleman and O. D. Lavrentovich, “Soft MatterPhysics: An Introduction,” Springer-Verlag New York, (2001). Thedirector n is in plane of the slab and depends on the in-planecoordinates (x,y). We assume that it does not depend on the verticalcoordinate z. The light beam impinges normally on the cell, along theaxis z. A polarizer placed between the source of light and the samplemakes the impinging light linearly polarized. In the nematic, thelinearly polarized wave of amplitude A intensity I₀=A² and the frequencyω splits into the ordinary and extraordinary waves with mutuallyperpendicular polarizations and amplitudes Asinβ and Acosβ,respectively; β(xy) is the angle between the local n(x,y) and thepolarization of incident light. The vibrations of the electric vectorsat the point of entry are in phase. However, the two waves takedifferent times, n_(o)d/c and n_(e)d/c, respectively, to pass throughthe slab. Here d is the thickness of the slab, and c is the speed oflight in vacuum. At the exit point, the electric vibrations${\sim A}\quad \sin \quad {\beta cos}\quad \left( {{\omega \quad t} - {\frac{2\pi}{\lambda_{0}}n_{o}d}} \right)\quad {\left. {and}\quad \right.\sim A}\quad \cos \quad \beta \quad \cos \quad \left( {{\omega \quad t} - {\frac{2\pi}{\lambda_{0}}n_{e}d}} \right)$

[0054] gain a phase shift${{\Delta \quad \phi} = {\frac{2\pi \quad d}{\lambda_{0}}\left( {n_{e} - n_{o}} \right)}},$

[0055] where λ₀ is the wavelength in vacuum. The projections of thesetwo vibrations onto the polarization direction of the analyzer behindthe sample are $\begin{matrix}{{a = {A\quad \sin \quad {\beta cos}\quad {\beta cos}\quad \left( {{\omega \quad t} - {\frac{2\pi}{\lambda_{0}}n_{o}d}} \right)\quad {and}}}{b = {{- A}\quad \sin \quad {\beta cos}\quad \beta \quad \cos \quad \left( {{\omega \quad t} - {\frac{2\pi}{\lambda_{0}}n_{e}d}} \right)}}} & \text{(Eq.~~1)}\end{matrix}$

[0056] When two harmonic vibrations A₁cos((ωt+Φ₁) and A₂cos(ωt+Φ₂) ofthe same frequency occur along the same directions, then the resultingvibration {overscore (A)}cos(ωt+{overscore (Φ)}) has an amplitudedefined from {overscore (A)}²=A₁ ²+A₂ ²+2A₁A₂cos(Φ₁−Φ₂). The analyzerthus transforms the pattern of (x,y)-dependent phase difference into thepattern of transmitted light intensity I(x,y)={overscore (A)}². Theintensity of light passed through the crossed polarizers and the nematicslab between them follows from Eq.(1) as $\begin{matrix}{I = {I_{0}\sin^{2}2\beta \quad {{\sin^{2}\left\lbrack {\frac{\pi \quad d}{\lambda_{0}}\left( {n_{e} - n_{o}} \right)} \right\rbrack}.}}} & \text{(Eq.~~2)}\end{matrix}$

[0057] The last formula refers to the case when n is perpendicular tothe axis z. If n makes an angle θ with the axis z, then (2) becomes$\begin{matrix}{I = {I_{0}\sin^{2}2\beta \quad {\sin^{2}\left\lbrack {\frac{\pi \quad d}{\lambda_{0}}\quad \left( {\frac{n_{o}n_{e}}{\sqrt{{n_{e}^{2}\cos^{2}\theta} + {n_{o}^{2}\sin^{2}\theta}}} - n_{o}} \right)} \right\rbrack}}} & \text{(Eq.~~3)}\end{matrix}$

[0058] Below is a representation of the propagation of light through apolarizer, uniaxial slab and analyzer.

[0059] The treatment can be further extended to describe the opticalproperties of complex director configurations, for example, in theelectric field-driven cells. However, for the case when the director isdistorted by ligand-receptor interactions rather than by an externallyapplied electric or magnetic field equations (2) and (3) are fundamentalfor understanding liquid crystal textures. First, note that the phaseshift and thus I depend on λ₀. As a result, when the sample isilluminated with a white light, it would show a colorful texture. Theinterference colors are especially pronounced when(n_(e)−n_(o))d˜(1÷3)λ₀. With typical (n_(e)−n_(o))˜0.2, λ₀˜500 nm, the‘colorful’ range of thicknesses is d˜(1÷10)μm. Second, the director tiltθ greatly changes the phase shift. When n∥z (the so-called homeotropicorientation, θ=0, the sample looks dark: only the ordinary wavepropagates and, according to Eq. (3), I=0. Third, if θ=0 but β=0, π/2, .. . , one might still observe dark textures, I=0, even innon-monochromatic light. In a sample with in-plane director distortionsn(x,y), wherever n (or its horizontal projection) is parallel orperpendicular to the polarizer, the propagating mode is either pureextraordinary or pure ordinary and the corresponding region of thetexture appears dark. By aligning a well-oriented liquid crystal samplebetween two crossed polarizers, one can find an “extinction” position inwhich the sample is dark. This extinction position corresponds to thedirector aligned along the polarization direction of polarizer oranalyzer, β=0, π/2, . . . , .

[0060] The extinction state will occur for all points of the sample, aslong as the director field is not perturbed and uniform. However, if thedirector field is disturbed and varies from point to point within theslab, then the condition of extinction (meaning I=0 in Equations (2) and(3)) cannot be satisfied everywhere and the resulting intensity of lightpassing through the polarizer, liquid crystal slab and analyser will bedifferent from zero. Such a disturbance of the liquid crystal detectorcan be caused by the receptor-ligand interaction, if this interactionrealigns the liquid crystalline molecules or aggregates in theneighborhood. These are the important features allowing us to use theliquid crystals as detection and amplification system.

[0061] Most biologic receptors possess both hydrophilic and hydrophobicregions and, thus, readily incorporate into biphilic lyotropic liquidcrystals. Additionally, the inactivated receptors do not destroy theoptical anisotropy (birefringence) of the liquid crystal and, therefore,the device comprised of a receptor-enriched liquid crystal with afollowing analyzer remains nontransparent to polarized light when properalignment satisfies the condition of extinction, as seen from equations(2) and (3). In this case, light would be able to pass through theliquid crystal but the analyzer would not let light pass any further,because the polarization of the light will be perpendicular to the planeof polarization of the analyzer. However, director orientation and,thus, the orientation of optical axis is disrupted when receptorconformation shifts as during the formation of the receptor-ligandcomplex. The elasticity of the liquid crystal enhances the localdistortions in the vicinity of the receptor-ligand complex, and expandsit to an optically detectable, supramicron scale. These distortionsgenerally deviate the director from the “extinction” orientations suchas β=0,±π/2, . . . , and make the system locally transparent, as thelight beam is not blocked by the analyzer.

Configurations of Ligand Detection Device

[0062] By way of example, one envisioned application of the presentinvention is in a multiwell system. Each well of the system wouldcontain PDAs to a specific ligand, such as a pathogenic microbe,interfaced with an amplification mechanism of the present invention.When the microbial agent interacts with the antibody, the resultingantibody distortion triggers the amplification mechanism. Preferably,the amplified signal is then transduced into a perceptible signal.Accordingly, it is envisioned that such a system could be placed in aphysician's -office, and be used in routine diagnostic procedures.Alternatively, such a system could be placed on or near soldiers inbattle, and the invention used to alert the soldiers to the presence ofa toxic agent. It is further envisioned that a multiwell system, ispreferably used in conjunction with the liquid crystal embodimentdescribed herein.

[0063] Thus, in one embodiment of the present invention, shownschematically in FIGS. 1B and 1C, a lyotropic liquid crystallinematerial is used as an amplification mechanism. As shown in FIG. 1B, thedevice consists of a light source 10, an initial polarizer 12, with thedirection of polarization in the plane of the figure, a pathogendetection system 14 a, comprising monospecific antibodies 14 b embeddedin biphilic, lyotropic liquid crystalline material 14 c, a secondarypolarizer 16, with the direction of polarization perpendicular to theplane of the figure, and a photodetector 18.

[0064] In operation, the initial polarizer 12 organizes a light beam 22that is linearly polarized in the plane of the figure. The optical axis20 of the inactivated device is perpendicular to the pathogen detectionsystem 14 a, and thus no birefringence of the transluminating linearlypolarized light stream 22 occurs. Since the polarization direction ofthe secondary polarizer 16 is perpendicular to the transluminatinglinearly polarized light 22, the secondary polarizer prevents light fromreaching the photodetector 18.

[0065] Binding of a ligand 24, such as a microbe, to the receptor 14 b,such as an antibody, distorts the liquid crystal 14 c, and thus causesdetectable changes in the light transmitted through the sample betweentwo crossed polarizers. This activation process is illustrated in FIG.1C. The receptor (antibody) 14 b is embedded in the lyotropic liquidcrystal 14 c. The spacial distortion caused by the formation of theantigen-antibody complex is transmitted to the contiguous liquid crystal14 c. The elastic characteristics of the liquid crystal permit thedistortion to be transmitted over a region much larger than the size ofthe receptor-ligand complex. This allows the use of the standard opticalphenomenon of birefringence to detect distortions caused by thereceptor-ligand complex, see Max Born and E. Wolf., Principals ofOptics, Sixth edition, Pressman Press, Oxford, 1980), as well as thediscussion above. The altered liquid crystalline order distorts theoptical axis and induces changes in the transmitted light, as discussedabove. For example, if the sample is originally aligned in the‘extinction” position (so that β0 or β=π/2), the transmission of lightthrough the two crossed polarizers and a sample between them is zero.The distortions caused by the receptor-ligand complex violate thecondition of complete extinction since these distortions deviate theangle β from the values β=0 and/or β=π/2. Therefore, the transmittanceof the light through the pair of polarizers and the liquid crystalsample will be different from zero in the regions of sample where thedistortions occur. The secondary polarizer (analyzer) 16 allows thisportion of light to pass to the photodetector 18. The detected change oramplification in light intensity can be transduced electronically into aperceptible signal.

[0066] In one preferred embodiment, the device of the present inventionmay include at least one substantially spherical substrate to whichreceptors may be attached. The receptor or receptors that are attachedto the spherical substrate must be capable of binding to a desiredligand to form a receptor-ligand complex such that, upon formation ofsaid receptor-ligand complex a signal is produced. An amplificationmechanism is interfaced with the receptor-ligand complex, where theamplification mechanism amplifies the signal produced by receptor-ligandcomplex formation.

[0067] The substantially spherical substrate utilized in the presentinvention can be non-porous (solid) or porous. In one embodiment, thesubstantially spherical substrate is a solid sphere and the at least onereceptor is attached to the outer surface of the spherical substrate.

[0068] In another embodiment, the substantially spherical substrate isporous. According to this embodiment, the at least one receptor may beattached to either the surface of said porous substantially sphericalsubstrate, the pores of said porous substantially spherical substrate,or both. By way of non-limiting example, if only one receptor isattached to the substantially spherical substrate, then the receptor canbe attached to either the outer surface of the porous sphere or in thepores of the sphere. In embodiment having more than one receptorattached to the spherical substrate, then the receptors can all beattached to the outer surface of the sphere, all the receptor can beattached within the pores of the sphere, or some receptors can beattached to the outer surface of the sphere and other receptors can beattached to the pores of the sphere. The use of a porous sphere or beadprovides a greater surface area on which to attach receptors and,therefore, would also permit surface and luminal receptor-ligandinteractions.

[0069] The receptors may be attached to the spherical substrate in anymanner known in the art, including chemical attachment and physicalattachment. In one preferred embodiment, the receptors are attached tothe spherical substrate by a chemical attachment, such as by covalentbonding to sulfate, amine, carboxyl or hydroxyl groups imbedded in thespherical substrate. However, it should be noted that the receptorswherein said at least one receptor is attached to said sphericalsubstrate by any means of physical attachment.

[0070] The substantially receptor-coated spherical substrate is madefrom a material including, but not limited to, polymeric and inorganicmaterials. In one preferred embodiment, the substantiallyreceptor-coated spherical substrate is comprised of a polymericmaterial. Suitable polymeric materials which may comprise the sphericalsubstrate include, but are not limited to, polyalkenes, polyacrylates,polymethacrylates, polyvinyls, polystyrenes, polycarbonates, polyesters,polyurethanes, polyamides, polyimides, polysulfones, polysiloxanes,polysilanes, polyethers, polycations, polyanions, and polycarboxylates.One particularly useful polymeric material used to manufacture thespherical substrate is polystyrene, especially when modified withcopolymers of acrylic ester, chloromethylstyrene, methylolamine, methylmethacrylate or made zwitterionic. If a polycation is utilized as thematerial of the spherical substrate, one particularly suitablepolycation is poly(diallyldimethylammoniumchloride).

[0071] In another embodiment, the substantially receptor-coatedspherical substrate is made from an inorganic material. Suitableinorganic materials include, but are not limited to, glass, silicon, andcolloidal gold. In one preferred embodiment, the spherical substrate isa glass bead.

[0072] The liquid crystalline material that is utilized with thesubstantially coated spherical substrate includes all known types ofthermotropic liquid crystalline materials and lyotropic liquidcrystalline materials. In one preferred embodiment, lyotropic liquidcrystalline material is used as the amplification mechanism. In anotherembodiment, lyotropic liquid crystalline materials of different origin,including surfactant and lyotropic chromonic liquid crystallinematerial, may used with the spherical substrate.

[0073] As described herein above, any receptor, such as antibodies orbiologic/biologically engineered receptors for ligands, can beincorporated into the device as long as binding of the ligand to thereceptor produces a detectable signal. Therefore, any type ofmonospecific antibody, including all polyclonal, monoclonal, or phagedisplayed antibodies can effectively function as a receptor.

[0074] In another embodiment, the present invention provides a methodfor detecting ligands. The method for detecting ligands, according thisembodiment, includes providing a device that comprises at least onesubstantially spherical substrate, at least one receptor attached tosaid spherical substrate, and an amplification mechanism. The at leastone receptor must be capable of binding to a ligand to form areceptor-ligand complex and, upon formation of the receptor-ligandcomplex, a signal is produced. The amplification mechanism must becapable of amplifying the signal produced by the receptor-ligand complexformation. Generally, a sample containing ligands specific to thereceptor that is attached to the sphere is exposed to the device. Afterexposing the ligand-containing sample to the device, the receptor orplurality of receptors that are attached to the sphere are allowed tointeract with the ligands in the sample to form at least onereceptor-ligand complex. The formation of the receptor-ligand complexproduces a detectable signal. The signal generated by the formation ofthe receptor-ligand complex is amplified by the amplification mechanism,namely, the liquid crystalline material. The amplified signal may thenbe measured and quantitated by those known methods easily determined bythose having ordinary skill in the art.

[0075] In one embodiment, the measurement and quantitation of the of thereceptor-ligand complex formation is mediated in the fluid phase or“flow through” phase, whereby the spheres and the liquid crystallinematerial are injected through an optical device that can determine theorientation of the liquid crystalline material. Utilizing thisparticular method of quantitation permits “field capture” of ligandsusing previously prepared spherical beads having a predeterminedreceptor attached thereto. Thus, for example, the ligands can becaptured “in the field”, transported, and analyzed at the later time.This method obviates the need for special transport media usuallyrequired to “protect” the ligand until detection is performed.

[0076] In another embodiment, the device for the detection of ligandscomprises at least one substantially spherical substrate coated with areceptor-binding or receptor-crosslinking material, at least onereceptor attached to the coated spherical substrate, and anamplification mechanism comprising a liquid crystalline material. The atleast one receptor is capable of binding to a ligand to form areceptor-ligand complex and the formation of the receptor-ligand complexproduces a signal. The signal produced is then amplified by theamplification mechanism upon receptor-ligand complex formation.According to the present embodiment, the crosslinker material may be,without limitation, natural or synthetic polymers, proteins, andsecondary antibodies.

[0077] In one preferred embodiment, molecules with specificity forreceptors, such as the specificity exhibited by Protein A, Protein G oranti-immunoglobulin antibodies for immunoglobulins, will be chemicallycross linked to the spherical substrate. Receptors with specificity forunique pathogens, toxins or proteins will then be bound to theimmobilized molecules.

[0078] In another embodiment, the present invention provides a methodfor detecting ligands comprising providing a device capable of detectingligands. According to this embodiment, the device comprises at least onesubstantially spherical substrate coated with a receptor-bindingmaterial; at least one receptor attached to said spherical substrate,and an amplification mechanism comprising a liquid crystalline material.The at least one receptor is capable of binding to a ligand to form areceptor-ligand complex and upon the formation of a receptor-ligandcomplex produces a signal. The amplification mechanism amplifies saidsignal upon receptor-ligand complex formation. The method includesexposing a sample containing at least one ligand to at least one of saidsubstrate and allowing the receptor to interact with the ligands in thesample to form at least one receptor-ligand complex. The signal producedby said receptor-ligand complex formation is then measured.

[0079] In another preferred embodiment, the device for detecting ligandscomprises an electrically charged, substantially planar substrate, atleast one receptor attached or bound to the planar substrate, and anamplification mechanism including a liquid crystalline material.

[0080] As described above the spherical substrates, the liquidcrystalline material that is utilized with the substantially coatedspherical substrate includes all known types of thermotropic liquidcrystalline materials and lyotropic liquid crystalline materials. In apreferred embodiment, lyotropic liquid crystalline materials are usedwith the electrically charged substrate. In another preferredembodiment, lyotropic chromonic liquid crystalline material is utilized.

[0081] In another embodiment, a method for detecting ligands isdisclosed comprising providing a device capable of detecting ligands,the device comprising at least one electrically charge substantiallyplanar substrate, at least one receptor attached to the substrate, andan amplification mechanism comprising a liquid crystalline material. Theat least one receptor is capable of binding to a ligand to form areceptor-ligand complex and the formation of a receptor-ligand complexproduces a signal. A sample containing ligands is exposed to thereceptor coated substrate, and is allowed to interact with the receptorsto form at least one receptor-ligand complex. The signal produced by thereceptor-ligand complex formation. is amplified by the liquidcrystalline amplification mechanism.

[0082] The present invention also provides a device for the detection ofligands including an electrically charged, substantially planarsubstrate, at least one receptor and an amplification mechanism. The atleast one receptor attached to the charged substrate is capable ofbinding to a ligand to form a receptor-ligand complex. The formation ofthe receptor-ligand complex produces a detectable signal, which isamplified by the amplification mechanism comprising a liquid crystallinematerial.

[0083] A charged substrate may be formed by depositing a polyionicmaterial from an aqueous solution onto the substrate. Withoutlimitation, for example, poly(diallyldimethylammoniumchloride) becomespositively charged in aqueous solutions as negatively charged Cl atomsdissociate from the molecule. To deposit the polyion layer onto a glasssubstrate, the substrate should be cleaned and then dipped it into theaqueous solution of the polyion. The polyion adsorbs to the surface ofthe substrate. The excess of the polyion can be washed out with anaqueous solution. In one preferred embodiment, an electrically chargedspherical substrate is utilized with lyotropic chromonic liquidcrystals. According to this embodiment, the opposite electric charges ofthe polyionic substrate and the chromonic liquid crystalline moleculesare kept in close contact by electrostatic forces.

[0084] In another embodiment, the present invention further provides adevice for the detection of ligands comprising an substantially planarsubstrate coated with a receptor-binding or crosslinking material, atleast one receptor, and an amplification mechanism. The at least onereceptor attached to the coated substrate is capable of binding to aligand to form a receptor-ligand complex. The formation of thereceptor-ligand complex produces a signal, which is amplified by theamplification mechanism comprising a liquid crystalline material. Asdescribed above for spherical substrates, the planar substrate is coatedwith molecules having specificity for receptors that include, withoutlimitation, polymers, Protein A, Protein G, anti-immunoglobulinantibodies for immunoglobulins. Receptors with specificity for uniquepathogens, toxins, or proteins will then be bound to the immobilizedreceptor-binding or crosslinker molecules coated on the surface of thesubstrate.

[0085] In a variation of the is embodiment, the coated substantiallyplanar substrate may also be electrically charged by any suitable means.

[0086] In one preferred embodiment, when utilizing any of the abovedescribed ligand detection and amplification devices, the non-specificaggregates are removed from the ligand containing sample prior toreacting the ligands with receptor and measuring the signal produced.The non-specific aggregates may be removed by any suitable meansincluding, but not limited to, filtering. The filtered sample will thenbe reacted with the desired receptor and the resulting signal producedby the formation of receptor-ligand complex will be amplified by theliquid crystalline material and measured. Without being bound to anyparticular theory, it is thought that the presence of the largenon-specific aggregates will increase light transmission through theliquid crystalline material and may, therefore, produce false positivesignals.

EXAMPLES

[0087] The following examples demonstrate the use of one embodiment ofthe present invention, namely, substantially receptor-coatedmicrospheres with the liquid crystal amplification mechanism to detectand amplify ligands upon receptor-ligand complex formation. A liganddetection system was created by introducing into the liquid crystalamplification mechanism a desired quantity of microspheres whose surfacewas substantially coated with microbe-specific antibodies. The examplesare intended for illustrative purposes only, and should not be construedas limiting the scope of the present invention in any manner.

[0088] The devices were evaluated by inserting antibody-coatedmicrospheres into a lyotropic liquid crystal. For each assay, 10 μl ofserially diluted microspheres (coated with either the anti-E. coli K99antibody or BSA) was mixed with 10 μl of the stock E. coli solution andincubated for 30 minutes at room temperature. The 20% stock solution ofliquid crystal (50 μl) was added to the microsphere-antibody solutionand gently mixed prevent the formation of bubbles. A 60 μl fraction ofthe mixture was deposited on a clean, polymer-coated glass square (1 mmthick; 25 mm square). A second cleaned, polymer-coated glass square wasaligned with the first square and pressure applied to uniformlydistribute the sample. A sample depth of approximately 20 μm wasmaintained by mylar spacers located between the two glass squares. Theedges of the glass assay chambers were sealed with nail polish.

[0089] Liquid crystals are anisometric molecules that exhibit limitedchemical interaction but that tend to orient along a common direction(the director). Director orientation is affected by externally appliedfields (electrical and magnetic); at the boundary between the liquidcrystal and it's container and flow. The liquid crystal orientation wasoptimized by constructing glass assay chambers that enhancedcontainer-liquid crystal interaction. The chambers were created asfollows: Borosilicate glass (1.0 mm thick; 200 mm×200 mm) plates werecleaned for 5 minutes in an 60° C., ultrasonic bath containing AlconoxDetergent (Fisher Scientific; Hanover Park, Ill.; product # 04-322-4) inwater, rinsed in distilled water and dried at 100° C. Each plate wascoated with an aligned layer of a polymer. The glass plate was cut into25 mm squares. A 25 mm square was positioned polymer up with two mylarspacer strips (20 μm thick, 2.0 mm×25 mm) located on the outer edges ofthe glass parallel to the orientation of the polymer. Liquidcrystal-microsphere samples (60 μl) were applied at the bottom edge ofthe glass between the mylar strips and a second 25 mm polymer-coatedglass was positioned so that it's polymer orientation was parallel tothe bottom glass. Pressure was applied to the top glass to distributethe sample. The edges were sealed with an appropriate sealing material.

[0090] Two liquid crystal solutions were evaluated. Lyotropic liquidcrystals were formed when either 20% disodium cromoglycate (Hartshorneand Woodard, Mol. Cryst. Liq. Cryst. 23:343, 1973) or 20% neutral greywas added to 80% distilled water (w/v). Preliminary phase diagramsdemonstrate that both the disodium cromoglycate (Sigma Chem. Co, St.Louis, Mo.; product # C0399) and the neutral grey (Optiva Inc., SanFrancisco, Calif.) liquid crystalline solutions remained in nematicphase at 24° C. when diluted to a 14% solution.

[0091] For Examples 1-8, cultures of E. coli (ATCC number 23503), grownto mid log growth phase in tryptic soy broth (Becton Dickinson, Sparks,Md.; product # 211822), were washed free of growth medium with twowashes of Phosphate Buffered Saline were used. The optical density ofeach E. coli suspension at 600 nm was measured and the bacteriaconcentration extrapolated from a growth curve (optical density at 600nm versus colony-forming units (CFUs)). Bacteria were then diluted withsterile phosphate buffered saline (PBS) to a concentration of 10⁸ CFUper 10 μl.

[0092] Each mixture was evaluated for light transmissive zones at200×magnification using a microscope equipped with crossed polarizers.For each assay cassette, the number of light transmissive zones in tenmicroscope fields were counted and the mean number per field calculated.Each experiment was conducted in duplicate. The data points in each ofthe following graphs represent the mean of the duplicate experiments.

Example 1

[0093] A commercially available 1.0 μm diameter polystyrene microspherewas obtained (Polysciences, Inc, Warrington, Pa.). The polystyrenemicrosphere was coated with a protein that tightly binds microbespecific antibodies. Protein G, a S. aureus protein that binds the Fcfraction of immunoglobulins, was cross-linked to the outer surface ofthe polystyrene microspheres.

[0094] A commercially available murine antibody (Accurate Chemical Co.;Westbury, N.Y.; product # YCC-311-603) specific to the sex pili (K99) ofE. coli bacteria was obtained and used undiluted. A stock solution ofassay microspheres (10⁷/μl) was created by incubating 44 μl ofmicrospheres with 56 μl of the murine anti-E. coli antibody for 30minutes at room temperature. The solution was washed twice withphosphate buffered saline to remove unbound primary antibody.

[0095] 10 μl of serially diluted polystyrene microspheres coated withthe anti-E. coli K99 antibody was mixed with 10 μl of the stock E. colisolution and incubated for 30 minutes at room temperature. The 20% stocksolution of neutral grey liquid crystal (50 μl) was added to themicrosphere-antibody solution, mixed, and the samples gently centrifuged(3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction of the mixturewas introduced into the glass assay chamber described above.

Comparative Example 2

[0096] A commercially available 1.0 μm diameter polystyrene microspherewas obtained (Polysciences, Inc, Warrington, Pa.). The polystyrenemicrosphere was coated with a protein that tightly binds microbespecific antibodies. Protein G, a S. aureus protein that binds the Fcfraction of immunoglobulins, was cross-linked to the outer surface ofthe polystyrene microspheres.

[0097] A stock solution of assay microspheres (10⁷/μl) was created byincubating 44 μl of microspheres with 56 μl BSA for 30 minutes at roomtemperature. The solution was washed twice with phosphate bufferedsaline to remove unbound primary antibody.

[0098] 10 μl of serially diluted polystyrene microspheres coated withBSA was mixed with 10 μl of the stock E. coli solution and incubated for30 minutes at room temperature. The 20% stock solution of the neutralgrey liquid crystal (50 μl) was added to the microsphere-antibodysolution, mixed, and the samples gently centrifuged (3500 g; 5 sec.) toeliminate bubbles. A 60 μl fraction of the mixture was introduced intothe glass assay chamber described above.

Example 3

[0099] A commercially available 1.0 μm diameter polycarboxylatemicrosphere was obtained (Polysciences, Inc, Warrington, Pa.). Thepolycarboxylate microsphere was coated with a protein that tightly bindsmicrobe specific antibodies. The polycarboxylate microsphere was coatedwith a goat immunoglobulin that binds all mouse immunoglobulins.

[0100] As described in Example 1 above, a commercially available murineantibody specific to the sex pili (K99) of E. coli bacteria was obtainedand used undiluted. A stock solution of assay microspheres (10⁷/μl) wascreated by incubating 44 μl of microspheres with 56 μl of the murineanti-E. coli antibody for 30 minutes at room temperature. The solutionwas washed twice with phosphate buffered saline to remove unboundprimary antibody.

[0101] 10 μl of serially diluted polycarboxylate microspheres coatedwith the anti-E. coli K99 antibody was mixed with 10 μl of the stock E.coli solution and incubated for 30 minutes at room temperature. The 20%stock solution of neutral grey liquid crystal (50 μl) was added to themicrosphere-antibody solution, mixed, and the samples gently centrifuged(3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction of the mixturewas introduced into the glass assay chamber described above.

Comparative Example 4

[0102] A commercially available 1.0 μm diameter polycarboxylatemicrosphere was obtained (Polysciences, Inc, Warrington, Pa.). Thepolycarboxylate microsphere was coated with a protein that tightly bindsmicrobe specific antibodies. The polycarboxylate microsphere was coatedwith a goat immunoglobulin that binds all mouse immunoglobulins.

[0103] A stock solution of assay microspheres (10⁷/μl) was created byincubating 44 μl of microspheres with 56 μl of BSA for 30 minutes atroom temperature. The solution was washed twice with phosphate bufferedsaline to remove unbound primary antibody.

[0104] 10 μl of serially diluted polycarboxylate microspheres coatedwith BSA was mixed with 10 /μl of the stock E. coli solution andincubated for 30 minutes at room temperature. The 20% stock solution ofthe neutral grey liquid crystal (50 μl) was added to themicrosphere-antibody solution, mixed, and the samples gently centrifuged(3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction of the mixturewas introduced into the glass assay chamber described above.

[0105] Example 5

[0106] A commercially available 1.0 μm diameter polystyrene microspherewas obtained (Polysciences, Inc, Warrington, Pa.). The polystyrenemicrosphere was coated with a protein that tightly binds microbespecific antibodies. Protein G, a S. aureus protein that binds the Fcfraction of immunoglobulins, was cross-linked to the outer surface ofthe polystyrene microspheres.

[0107] A commercially available murine antibody (Accurate Chemical Co.;Westbury, N.Y.; product # YCC-311-603) specific to the sex pili (K99) ofE. coli bacteria was obtained and used undiluted. A stock solution ofassay microspheres (10⁷/μl) was created by incubating 44 μl ofmicrospheres with 56 μl of the murine anti-E. coli antibody for 30minutes at room temperature. The solution was washed twice withphosphate buffered saline to remove unbound primary antibody.

[0108] 10 μl of serially diluted polystyrene microspheres coated withthe anti-E. coli K99 antibody was mixed with 10 μl of the stock E. colisolution and incubated for 30 minutes at room temperature. The 20% stocksolution of disodium cromoglycate liquid crystal (50 μL) was added tothe microsphere-antibody solution, mixed, and the samples gentlycentrifuged (3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction ofthe mixture was introduced into the glass assay chamber described above.

[0109] Comparative Example 6

[0110] A commercially available 1.0 μm diameter polystyrene microspherewas obtained (Polysciences, Inc, Warrington, Pa.). The polystyrenemicrosphere was coated with a protein that tightly binds microbespecific antibodies. Protein G, a S. aureus protein that binds the Fcfraction of immunoglobulins, was cross-linked to the outer surface ofthe polystyrene microspheres.

[0111] A stock solution of assay microspheres (10⁷/μl) was created byincubating 44 μl of microspheres with 56 μl BSA for 30 minutes at roomtemperature. The solution was washed twice with phosphate bufferedsaline to remove unbound primary antibody.

[0112] 10 μl of serially diluted polystyrene microspheres coated withBSA was mixed with 10 μl of the stock E. coli solution and incubated for30 minutes at room temperature. The 20% stock solution of the disodiumcromoglycate liquid crystal (50 μl) was added to themicrosphere-antibody solution, mixed, and the samples gently centrifuged(3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction of the mixturewas introduced into the glass assay chamber described above.

[0113] Example 7

[0114] A commercially available 1.0 μm diameter polycarboxylatemicrosphere was obtained (Polysciences, Inc, Warrington, Pa.). Thepolycarboxylate microsphere was coated with a protein that tightly bindsmicrobe specific antibodies. The polycarboxylate microsphere was coatedwith a goat immunoglobulin that binds all mouse immunoglobulins.

[0115] As described in Example 1 above, a commercially available murineantibody specific to the sex pili (K99) of E. coli bacteria was obtainedand used undiluted. A stock solution of assay microspheres (10⁷/μl) wascreated by incubating 44 μl of microspheres with 56 μl of the murineanti-E. coli antibody for 30 minutes at room temperature. The solutionwas washed twice with phosphate buffered saline to remove unboundprimary antibody.

[0116] 10 μl of serially diluted polycarboxylate microspheres coatedwith the anti-E. coli K99 antibody was mixed with 10 μl of the stock E.coli solution and incubated for 30 minutes at room temperature. The 20%stock solution of disodium cromoglycate liquid crystal (50 μl) was addedto the microsphere-antibody solution, mixed, and the samples gentlycentrifuged (3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction ofthe mixture was introduced into the glass assay chamber described above.

Comparative Example 8

[0117] A commercially available 1.0 μm diameter polycarboxylatemicrosphere was obtained (Polysciences, Inc, Warrington, Pa). Thepolycarboxylate microsphere was coated with a protein that tightly bindsmicrobe specific antibodies. The polycarboxylate microsphere was coatedwith a goat immunoglobulin that binds all mouse immunoglobulins.

[0118] A stock solution of assay microspheres (10⁷/μl) was created byincubating 44 μl of microspheres with 56 μl of BSA for 30 minutes atroom temperature. The solution was washed twice with phosphate bufferedsaline to remove unbound primary antibody.

[0119] 10 μl of serially diluted polycarboxylate microspheres coatedwith BSA was mixed with 10 μl of the stock E. coli solution andincubated for 30 minutes at room temperature. The 20% stock solution ofthe disodium chromoglycate liquid crystal (50 μl) was added to themicrosphere-antibody solution, mixed, and the samples gently centrifuged(3500 g; 5 sec.) to eliminate bubbles. A 60 μl fraction of the mixturewas introduced into the glass assay chamber described above.

[0120] Ligand (bacteria)-bound microsphere aggregates distorted theliquid crystal director to cause local zones of light transmission,which were easily detected.

[0121]FIG. 4A demonstrates that increasing numbers of light transmissivezones occur in a 14% neutral grey liquid crystalline solution as theratio of E. coli to polycarboxylate microspheres increases.

[0122]FIG. 4B demonstrates that increasing numbers of light transmissivezones occur in a 14% neutral grey liquid crystalline solution as theratio of E. coli to polystyrene microspheres increases.

[0123]FIG. 5A shows that increasing numbers of light transmissive zonesoccur in a 14% disodium cromoglycate liquid crystalline solution as theratio of E. coli to polycarboxylate microspheres increases.

[0124]FIG. 5B shows that increasing numbers of light transmissive zonesoccur in a 14% disodium cromoglycate liquid crystalline solution as theratio of E. coli to polystyrene microspheres increases.

[0125] Greater light transmission occurred at microsphere to E. coliratios exceeding 1:4. In all experiments, antibody-coated microspheresinduced the formation of more light transmissive zones than did thecontrol microspheres coated with Bovine Serum Albumin.

[0126] It is to be understood that any variations evident fall withinthe scope of the claimed invention, and thus the selection of specificreceptors, such as antibodies and liquid crystals can be determinedwithout departing from the spirit of the invention herein disclosed anddescribed. It should also be understood that the present invention,while particularly suited for pathogen detection, is intended to includethe detection of any ligand. Moreover, the scope of the invention shallinclude all modifications and variations that may fall within the scopeof the attached claims.

We claim:
 1. A device for the detection of ligands comprising: at leastone substantially spherical substrate; at least one receptor attached tosaid spherical substrate, wherein said at least one receptor is capableof binding to a ligand to form a receptor-ligand complex and wherein theformation of said receptor-ligand complex produces a signal; and anamplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation.
 2. The device of claim 1, whereinsaid substantially spherical substrate is non-porous.
 3. The device ofclaim 2, wherein said at least one receptor is attached to the surfaceof said non-porous substantially spherical substrate.
 4. The device ofclaim 1, wherein the substantially spherical substrate is porous.
 5. Thedevice of claim 4, wherein said at least one receptor is attached to atleast one of (i) the surface of said porous substantially sphericalsubstrate and (ii) the pores of said porous substantially sphericalsubstrate.
 6. The device of claim 5, wherein said at least one receptoris attached to the surface of said porous substantially sphericalsubstrate.
 7. The device of claim 5, wherein said at least one receptoris attached to the pores of said porous substantially sphericalsubstrate.
 8. The device of claim 5, wherein a plurality of receptorsare attached to and randomly distributed on the surface and within thepores of said porous substantially spherical substrate.
 9. The device ofclaim 1, wherein the liquid crystalline material is selected from thegroup consisting of thermotropic liquid crystalline material andlyotropic liquid crystalline material.
 10. The device of claim 9,wherein the liquid crystalline material is a lyotropic liquidcrystalline material.
 11. The device of claim 10, wherein the lyotropicliquid crystalline material is a lyotropic chromonic liquid crystallinematerial.
 12. The device of claim 9, wherein the liquid crystallinematerial is a thermotropic liquid crystalline material.
 13. The deviceof claim 1, wherein the substantially spherical substrate is made from amaterial selected from the group consisting of polymeric and inorganicmaterials.
 14. The device of claim 13, wherein the substantiallyspherical substrate is made from a polymeric material.
 15. The device ofclaim 14, wherein the polymeric materials are selected from the groupconsisting of polyions, polyalkenes, polyacrylates, polymethacrylates,polyvinyls, polystyrenes, polycarbonates, polyesters, polyurethanes,polyamides, polyimides, polysulfones, polysiloxanes, polysilanes,polyethers, and polycarboxylates.
 16. The device of claim 15, whereinthe polymeric material is a polystyrene.
 17. The device of claim 13,wherein the substantially spherical substrate is made from an inorganicmaterial.
 18. The device of claim 17, wherein the inorganic material isselected from the group consisting of glass, silicon, and colloidalgold.
 19. The device of claim 18, wherein the inorganic material isglass.
 20. The device of claim 1, wherein said at least one receptor isattached to said spherical substrate by one means selected from thegroup consisting of (i) chemical attachment and (ii) physicalattachment.
 21. The device of claim 20, wherein said at least onereceptor is attached to said spherical substrate by chemical attachment.22. The device of claim 21, wherein said chemical attachment is covalentbonding.
 23. The device of claim 20, wherein said at least one receptoris attached to said spherical substrate by physical attachment.
 24. Themethod of claim 23, wherein said physical attachment is selected fromthe group consisting hydrophobic interactions and van der Waalsinteractions.
 25. A method for detecting ligands comprising: providing adevice capable of detecting ligands, said device comprising at least onesubstantially spherical substrate; at least one receptor attached tosaid spherical substrate, wherein said at least one receptor is capableof binding to a ligand to form a receptor-ligand complex and wherein theformation of said receptor-ligand complex produces a signal; and anamplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation; exposing a sample containing at leastone ligand to said at least one substrate; allowing said receptor tointeract with said at least one ligand to form at least onereceptor-ligand complex; and measuring the signal produced by saidreceptor-ligand complex formation.
 26. A device for the detection ofligands comprising: at least one substantially spherical substratecoated with a receptor-binding material; at least one receptor attachedto said coated spherical substrate, wherein said at least one receptoris capable of binding to a ligand to form a receptor-ligand complex andwherein the formation of said receptor-ligand complex produces a signal;and an amplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation.
 27. The device of claim 26, whereinsaid substantially spherical substrate is non-porous.
 28. The device ofclaim 27, wherein said at least one receptor is attached to the surfaceof said non-porous substantially spherical substrate.
 29. The device ofclaim 26, wherein the substantially spherical substrate is porous. 30.The device of claim 29, wherein said at least one receptor is attachedto at least one of (i) the surface of said porous substantiallyspherical substrate and (ii) the pores of said porous substantiallyspherical substrate.
 31. The device of claim 30, wherein said at leastone receptor is attached to the surface of said porous substantiallyspherical substrate.
 32. The device of claim 30, wherein said/at leastone receptor is attached to the pores of said porous substantiallyspherical substrate.
 33. The device of claim 30, wherein a plurality ofreceptors are attached to and randomly distributed on the surface andwithin the pores of said porous substantially spherical substrate. 34.The device of claim 26, wherein the liquid crystalline material isselected from the group consisting of thermotropic liquid crystallinematerial and lyotropic liquid crystalline material.
 35. The device ofclaim 34, wherein the liquid crystalline material is a lyotropic liquidcrystalline material.
 36. The device of claim 35, wherein the lyotropicliquid crystalline material is a lyotropic chromonic liquid crystallinematerial.
 37. The device of claim 34, wherein the liquid crystallinematerial is a thermotropic liquid crystalline material.
 38. The deviceof claim 26, wherein the substantially spherical substrate is made froma material selected from the group consisting of polymeric and inorganicmaterials.
 39. The device of claim 38, wherein the substantiallyspherical substrate is made from a polymeric material.
 40. The device ofclaim 39, wherein the polymeric materials are selected from the groupconsisting of polyions, polyalkenes, polyacrylates, polymethacrylates,polyvinyls, polystyrenes, polycarbonates, polyesters, polyurethanes,polyamides, polyimides, polysulfones, polysiloxanes, polysilanes,polyethers, and polycarboxylates.
 41. The device of claim 40, whereinthe polymeric material is a polystyrene.
 42. The device of claim 38,wherein the substantially spherical substrate is made from an inorganicmaterial.
 43. The device of claim 42, wherein the inorganic material isselected from the group consisting of glass, silicon, and colloidalgold.
 44. The device of claim 43, wherein the inorganic material isglass.
 45. The device of claim 26, wherein said at least one receptor isattached to said spherical substrate by one means selected from thegroup consisting of (i) chemical attachment and (ii) physicalattachment.
 46. The device of claim 45, wherein said at least onereceptor is attached to said spherical substrate by chemical attachment.47. The device of claim 46, wherein said chemical attachment is covalentbonding.
 48. The device of claim 45, wherein said at least one receptoris attached to said spherical substrate by physical attachment.
 49. Themethod of claim 48, wherein said physical attachment is selected fromthe group consisting hydrophobic interactions and van der Waalsinteractions.
 50. A method for detecting ligands comprising: providing adevice capable of detecting ligands, said device comprising at least onesubstantially spherical substrate coated with a receptor-bindingmaterial; at least one receptor attached to said spherical substrate,wherein said at least one receptor is capable of binding to a ligand toform a receptor-ligand complex and wherein the formation of saidreceptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation; exposing a sample containing at least one ligand toat least one of said substrate; allowing said receptor to interact withsaid at least one ligand to form at least one receptor-ligand complex;and measuring the signal produced by said receptor-ligand complexformation.
 51. A device for the detection of ligands comprising: asubstantially planar substrate, wherein said substrate is electricallycharged; at least one receptor attached to said electrically chargedsubstantially planar substrate, wherein said at least one receptor iscapable of binding to a ligand to form a receptor-ligand complex andwherein the formation of said receptor-ligand complex produces a signal;and an amplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation.
 52. The device of claim 51, whereinthe liquid crystalline material is selected from the group consisting ofthermotropic liquid crystalline material and lyotropic liquidcrystalline material.
 53. The device of claim 52, wherein the liquidcrystalline material is a lyotropic liquid crystalline material.
 54. Thedevice of claim 53, wherein the lyotropic liquid crystalline material isa lyotropic chromonic liquid crystalline material.
 55. The device ofclaim 52, wherein the liquid crystalline material is a thermotropicliquid crystalline material.
 56. The device of claim 51, wherein thesubstantially planar substrate is made from a material selected from thegroup consisting of polymeric and inorganic materials.
 57. The device ofclaim 56, wherein the substantially planar substrate is made from apolymeric material.
 58. The device of claim 57, wherein the polymericmaterials are selected from the group consisting of polyions,polyalkenes, polyacrylates, polymethacrylates, polyvinyls, polystyrenes,polycarbonates, polyesters, polyurethanes, polyamides, polyimides,polysulfones, polysiloxanes, polysilanes, polyethers, andpolycarboxylates.
 59. The device of claim 58, wherein the polymericmaterial is a polystyrene.
 60. The device of claim 56, wherein thesubstantially planar substrate is made from an inorganic material. 61.The device of claim 60, wherein the inorganic material is selected fromthe group consisting of glass, silicon, and colloidal gold.
 62. Thedevice of claim 61, wherein the inorganic material is glass.
 63. Thedevice of claim 51, wherein said at least one receptor is attached tosaid substrate by one means selected from the group consisting of (i)chemical attachment and (ii) physical attachment.
 64. The device ofclaim 63, wherein said at least one receptor is attached to saidspherical substrate by chemical attachment.
 65. The device of claim 64,wherein said chemical attachment is covalent bonding.
 66. The device ofclaim 63, wherein said at least one receptor is attached to saidspherical substrate by physical attachment.
 67. The method of claim 66,wherein said physical attachment is selected from the group consistinghydrophobic interactions and van der Waals interactions.
 68. A methodfor detecting ligands comprising: providing a device capable ofdetecting ligands, said device comprising at least one electricallycharge substantially planar substrate; at least one receptor attached tosaid substrate, wherein said at least one receptor is capable of bindingto a ligand to form a receptor-ligand complex and wherein the formationof said receptor-ligand complex produces a signal; and an amplificationmechanism comprising a liquid crystalline material, wherein saidamplification mechanism amplifies said signal upon receptor-ligandcomplex formation; exposing a sample containing at least one ligand tosaid substrate; allowing said receptor to interact with said at leastone ligand to form at least one receptor-ligand complex; and measuringthe signal produced by said receptor-ligand complex formation.
 69. Adevice for the detection of ligands comprising: an substantially planarsubstrate coated with a receptor-binding material; at least one receptorattached to said substrate, wherein said at least one receptor iscapable of binding to a ligand to form a receptor-ligand complex andwherein the formation of said receptor-ligand complex produces a signal;and an amplification mechanism comprising a liquid crystalline material,wherein said amplification mechanism amplifies said signal uponreceptor-ligand complex formation.
 70. The device of claim 69, whereinthe liquid crystalline material is selected from the group consisting ofthermotropic liquid crystalline material and lyotropic liquidcrystalline material.
 71. The device of claim 70, wherein the liquidcrystalline material is a lyotropic liquid crystalline material.
 72. Thedevice of claim 71, wherein the lyotropic liquid crystalline material isa lyotropic chromonic liquid crystalline material.
 73. The device ofclaim 70, wherein the liquid crystalline material is a thermotropicliquid crystalline material.
 74. The device of claim 69, wherein thesubstrate is made from a material selected from the group consisting ofpolymeric and inorganic materials.
 75. The device of claim 74, whereinthe substrate is made from material a polymeric material.
 76. The deviceof claim 75, wherein the polymeric materials are selected from the groupconsisting of polyalkenes, polyacrylates, polymethacrylates, polyvinyls,polystyrenes, polycarbonates, polyesters, polyurethanes, polyamides,polyimides, polysulfones, polysiloxanes, polysilanes, polyethers, andpolycarboxylates.
 77. The device of claim 76, wherein the polymericmaterial is polystyrene.
 78. The device of claim 74, wherein thesubstantially substrate is made from an inorganic material.
 79. Thedevice of claim 78, wherein the inorganic material is selected from thegroup consisting of glass, silicon, and colloidal gold.
 80. The deviceof claim 79, wherein the inorganic material is glass.
 81. The device ofclaim 69, wherein said at least one receptor is attached to saidsubstrate by one means selected from the group consisting of (i)chemical attachment and (ii) physical attachment.
 82. The device ofclaim 80, wherein said at least one receptor is attached to saidsubstrate by chemical attachment.
 83. The device of claim 81, whereinsaid chemical attachment is covalent bonding.
 84. The device of claim80, wherein said at least one receptor is attached to said substrate byphysical attachment.
 85. The method of claim 84, wherein said physicalattachment is selected from the group consisting of hydrophobicinteractions and van der Waals interactions.
 86. The device of claim 69,wherein the coated substantially planar substrate is electricallycharged.
 87. A method for detecting ligands comprising: providing adevice capable of detecting ligands, said device comprisingsubstantially planar substrate coated with a receptor-binding material;at least one receptor attached to said substrate, wherein said at leastone receptor is capable of binding to a ligand to form a receptor-ligandcomplex and wherein the formation of said receptor-ligand complexproduces a signal; and an amplification mechanism comprising a liquidcrystalline material, wherein said amplification mechanism amplifiessaid signal upon receptor-ligand complex formation; exposing a samplecontaining at least one ligand to said substrate; allowing said receptorto interact with said at least one ligand to form at least onereceptor-ligand complex; and measuring the signal produced by saidreceptor-ligand complex formation.
 88. The method of claim 87, whereinthe coated substantially planar substrate is electrically charged.